Retinitis pigmentosa 25 (RP25) is a common autosomal recessive genetic disease caused by abnormal eyes shut homolog protein (EYS). There is, however, little understanding of the mechanism by which mutations to EYS cause RP25 and there is no effective therapy. One of the challenges in developing a mechanistic un- derstanding of EYS function is the fact that the mouse does not have the EYS gene. To overcome this signifi- cant hurdle, we have developed a zebrafish model for RP25. Zebrafish express the EYS gene. EYS-deficient zebrafish show age-dependent photoreceptor degeneration similar to patients with RP25. Therefore, the goal of this project is to decipher the mechanisms of retinal degeneration caused by mutations in EYS using the zebrafish model system. EYS is a secreted protein containing multiple epidermal growth factor (EGF) and laminin globular (LG) domains. The latter is a conserved domain structure that is capable of binding to O- mannosyl glycans of ?-dystroglycan, a cell surface receptor for extracellular matrix. Preliminary data indicate that the EYS protein binds to O-mannosyl glycans and is co-localized with the tectonic complex near the con- necting cilium/transition zone in photoreceptor cells. Abnormal O-mannosyl glycosylation underlie syndromic retinal atrophy found in congenital muscular dystrophies with brain malformations. Interestingly, hypomorphic mutations in one of the enzymes involved in O-mannosyl glycosylation are found in retinitis pigmentosa 76 (RP76). Tectonic complex proteins are implicated in severe ciliopathies such as Joubert syndrome. Our cen- tral hypothesis is that EYS is a key member of the ciliary pocket matrix interacting with O-mannosyl glycans and the tectonic complex at the connecting cilium/transition zone. Aim 1: Determine the roles of EYS in maintaining photoreceptor health. Aim 2: Determine the roles of O-mannosyl glycans in photoreceptor survival. Aim 3: Characterize EYS interactions with the tectonic complex at connecting cilium/transition zone. Multidisciplinary approaches including immuno-EM, cryo-EM, immunohistological staining, genetic manipu- lations in zebrafish embryos, and biochemical experiments such as co-immunoprecipitation, proximity ligation assay, and proteomic methods will be employed to accomplish the aims. Together, the proposed research will provide insights on molecular pathogenic mechanisms of the disease, uncover how EYS regulates ciliary func- tions in photoreceptors, and reveal the proteins that interact with EYS at the ciliary pocket. It has the potential to link several forms of retinal degeneration into a common biological pathway where cell-extracellular matrix interactions mediated by EYS help maintain the ciliary pocket structural integrity to maintain photoreceptor survival. We, thus, will have a better understanding of the mechanisms of photoreceptor degeneration caused by abnormal EYS, which will provide new opportunities to develop therapeutic interventions.